The recent advances in genetic engineering and biotechnology have placed the chemical process industry at the doorstep of a new era of products never before commercially available. The production of human insulin on a commercial basis has already begun and other pharmaceuticals are certain to follow as processes are developed for producing these highly complex molecules which may have a profound impact on our ability to treat disease and metabolic abnormalities. Pesticides, fertilizers and fine chemicals will also be likely candidates for new biotechnology applications.
As the new biotechnological processes are developed and scaled-up to production levels, the separation and purification of products will be a critical factor in determining the technical feasibility as well as the economic success of the process. Indeed, the second largest cost associated with producing products from biological processes is the cost of the product separation and purification.
Distillation and extraction are the most widely used methods in the petrochemical industry; however, these methods are generally unsuitable for bioseparations. Many biochemically active compounds are active only over specific ranges of temperature, pH, and ionic strength. The molecule may also be affected by interaction with the solvent. These conformational changes are not necessarily reversible so great care must be taken to ensure mild operating conditions and thus avoid degradation of the valuable product during the recovery phase. Furthermore, fermentation broths are typically quite dilute compared to other chemical process streams, and contain a complex mixture of substances some of which are closely related to the desired product.
Due to these rather severe limitations, membrane processes, ultra-centrifugation, differential precipitation, electrophoresis and chromatography are the techniques used most often in bench scale procedures. These procedures are usually performed batchwise and have very small product throughputs--milligram quantities are not uncommon as an upper limit.
Electrophoresis and chromatography are the two most useful techniques to modern chemical analysis. They have the highest resolution and can be quite highly specific when used in conjunction with affinity or immunochemical techniques. Electrophoresis is a particularly attractive technique since it has high resolution capability and would be most useful in the final purification of products. Furthermore, since it separates principally on the basis of charge rather than size, it is well-suited for the separation of similarly sized charged molecules from one another as well as from uncharged molecules.
The problems involved in the implementation of an electrophoresis process capable of industrial-scale use are not in the physical separation of products, but rather in the practical engineering design of the electrophoretic device. Such a device must provide for (1) the continuous supply of both elutant and feed to the bed, (2) the continuous removal of products from the bed, (3) the efficient removal of heat from the bed to limit the bed temperature rise, (4) large throughput, and (5) a large number of purified product fractions. A search of the prior art revealed the following:
Vermeulen and colleagues ("Design Theory and Separations in Preparative-Scale Continuous-Flow Annular-Bed Electrophoresis", Ind. Eng. Chem. Process Des. Develop., 10:91-102 (1971)) have developed a continuous flow electrophoretic separator. It separates product in the radial direction and is limited in the number of products. Furthermore, the heating which takes place causes a substantial temperature rise.
Nerenburg (U.S. Pat. No. 3,704,217) describes a batch or non-continuous electrophoretic separation device whereby a batch sample is introduced in a packed column and the current is turned on to separate the sample into various components. The current is then turned off and the various products are found to be distributed along the length of the bed. A series of ports along the bed provide a means for washing out a given product at an intermediate location and eliminating the need for the product to traverse the entire length of the column.
Anderson (U.S. Pat. No. 3,556,967) describes a batch or non-continuous electrophoretic separation device whereby a density gradient is established in a rapidly spinning centrifuge rotor. The batch sample is introduced into this density gradient and the current is turned on. Separation takes place and mixing due to thermal heating is minimized as a result of the density gradient coupled with the centrifugal field. After the separation is complete the current is turned off. The density gradient and the separated products are then pumped out.
Bier (U.S. Pat. No. 4,040,940) describes a batch or non-continuous electrophoretic separation device which employs a rotational seal fraction collector. The actual separation device uses the method of rotationally stabilized flow in the annulus between horizontal concentric cylinders. The operation of this device requires that the annulus first be filled with elutant. The batch sample is introduced into the elutant stream. At this time, the rotation is started and the current turned on. As the separation takes place under the influence of the electric field, the product bands move toward the collector by the flow of elutant. At the collector, a small jet perpendicular to the flow forces the product out.
Fox, et al., ("Continuous Chromatography Apparatus, Part I, Construction," J. Chromatog., 43: 48-54 (1969); "Continuous Chromatography Apparatus, Part II, Operation" 43: 55-60 (1960); and "Continuous Chromatography Apparatus, Part III, Application," 43: 61-65 (1960)) and Begovich, et al., ("Multicomponent Separations Using a Continuous Annular Chromatograph," Ph.D. Dissertation, University of Tennessee, Knoxville, TN. (1982); "A Rotating Annular Chromatograph for Continuous Separations," Submitted to AlChE J.) have demonstrated the use of a slowly rotating annular bed of sorbent material to effect continuous chromatographic multicomponent separations. The rotation of the bed causes the separated components to appear as helical bands, each of which has a characteristic stationary exit point at some angular coordinate at the bottom of the column.
Note that in electrophoresis, actual separation only takes place while the electric current is on. Furthermore, a continuous process is one whereby the elutant and sample are introduced continuously into the device. Thus, the need for a continuous electrophoretic separation device which has a continual supply of elutant, sample or feed and electric current to maintain separation in the device at all times is required. Previous devices are not fully continuous and therefore, require that the device be operated in a cyclical fashion. At some point in the operation, the device must cease performing its separation function in order for it to be returned to some initial condition in order to receive a new sample for separation.
Although continuous electrophoresis is industrially attractive and feasible, for the full scale commercialization of recent advances in biotechnology, separation processes must be developed and refined to allow continuous operation and higher throughputs while still observing the restrictions mentioned previously to maintain bioactivity of the product.
Those concerned with these and other problems recognize the need for an improved apparatus for the separation of binary and multi-component mixtures of products from biological processes.